The major goal of this proposal is to use evolutionary protein design to explore the molecular mechanism(s) supporting H2O2-mediated oxygenation in heme-enzymes. Cytochrome P450 and chioroperoxidase (CPO) are both heme containing oxidoreductases that stereospecifically catalyze the H2O2-mediated oxygenation of organic substrates. These proteins, however, differ significantly in their catalytic efficiencies. It is not known why CPO catalyzes this reaction more efficiently than P450s and unclear whether a P450 scaffold is compatible with CPO-like activity. To better understand the molecular determinants that allow heme-enzymes to function efficiently, I plan to: 1) engineer P450 variants with CPO-like catalytic efficiency through directed evolution; 2) analyze the mechanism(s) by which P450 variants catalyze oxygenation; and 3) determine the structure of interesting mutants. Through comparison of the activities and structures of wild type and evolved P450 enzymes, I will determine whether P450s adapt a catalytic cycle and active site structure similar to CPO, or if they adapt properties distinct from known heme-enzymes. In addition to providing insight into heme-enzyme activity adaptation, these studies could also aid in the development of single-enzyme monooxygenases with sufficient activity to make them practical biocatalysts for medical and pharmaceutical applications.